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Neutrophil-Specific Granule Deficiency Leads to Ε Zipper A Novel In-Frame Deletion in the Leucine Zipper Domain of C/EBP ε Leads to Neutrophil-Specific Granule Deficiency This information is current as Taizo Wada, Tadayuki Akagi, Masahiro Muraoka, Tomoko of October 1, 2021. Toma, Kenzo Kaji, Kazunaga Agematsu, H. Phillip Koeffler, Takashi Yokota and Akihiro Yachie J Immunol 2015; 195:80-86; Prepublished online 27 May 2015; doi: 10.4049/jimmunol.1402222 http://www.jimmunol.org/content/195/1/80 Downloaded from Supplementary http://www.jimmunol.org/content/suppl/2015/05/27/jimmunol.140222 Material 2.DCSupplemental http://www.jimmunol.org/ References This article cites 29 articles, 18 of which you can access for free at: http://www.jimmunol.org/content/195/1/80.full#ref-list-1 Why The JI? Submit online. • Rapid Reviews! 30 days* from submission to initial decision by guest on October 1, 2021 • No Triage! Every submission reviewed by practicing scientists • Fast Publication! 4 weeks from acceptance to publication *average Subscription Information about subscribing to The Journal of Immunology is online at: http://jimmunol.org/subscription Permissions Submit copyright permission requests at: http://www.aai.org/About/Publications/JI/copyright.html Email Alerts Receive free email-alerts when new articles cite this article. Sign up at: http://jimmunol.org/alerts The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2015 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology A Novel In-Frame Deletion in the Leucine Zipper Domain of C/EBP« Leads to Neutrophil-Specific Granule Deficiency Taizo Wada,*,1 Tadayuki Akagi,†,1 Masahiro Muraoka,* Tomoko Toma,* Kenzo Kaji,‡ Kazunaga Agematsu,x H. Phillip Koeffler,{,‖ Takashi Yokota,† and Akihiro Yachie* Neutrophil-specific granule deficiency (SGD) is a rare autosomal recessive primary immunodeficiency characterized by neutrophil dysfunc- tion, bilobed neutrophil nuclei and lack of neutrophil-specific granules. Defects in a myeloid-specific transcription factor, CCAAT/enhancer binding protein-« (C/EBP«), have been identified in two cases in which homozygous frameshift mutations led to loss of the leucine zipper domain. In this study, we report a 55-y-old woman affected with SGD caused by a novel homozygous 2-aa deletion (DRS) in the leucine zipper domain of the C/EBP« gene. The patient showed characteristic neutrophil abnormalities and recurrent skin infections; however, D there was no history of deep organ infections. Biochemical analysis revealed that, in contrast to the two frameshift mutations, the RS Downloaded from mutant maintained normal cellular localization, DNA-binding activity, and dimerization, and all three mutants exhibited marked reduction in transcriptional activity. The DRS mutant was defective in its association with Gata1 and PU.1, as well as aberrant cooperative transcrip- tional activation of eosinophil major basic protein. Thus, the DRS likely impairs protein-protein interaction with other transcription factors, resulting in a loss of transcriptional activation. These results further support the importance of the leucine zipper domain of C/EBP« for its essential function, and indicate that multiple molecular mechanisms lead to SGD. The Journal of Immunology, 2015, 195: 80–86. http://www.jimmunol.org/ eutrophil-specific granule deficiency (SGD) is a rare au- in elevated levels of C/EBPε (4). In this patient, the growth fac- tosomal recessive primary immunodeficiency characterized tor independence 1 (Gfi-1) protein that represses transcription of N by either profound reduction or absence of neutrophil- C/EBPε was decreased, although the patient had no mutation of specific granules and bilobed neutrophil nuclei (pseudo–Pelgar- the Gfi-1 gene. Taken together, these previous findings suggest Hue¨t anomaly) (1). It was previously called “lactoferrin deficiency.” that SGD is a genetically heterogeneous disease. Because of the Patients with SGD exhibit increased susceptibility to bacterial in- extreme rarity of SGD, the full spectrum of clinical symptoms and fections, especially affecting the skin, ears, lungs, and lymph nodes. cellular abnormalities of the disease is unclear. The gene responsible for SGD is the CCAAT/enhancer binding C/EBPε is a member of the C/EBP family of widely expressed protein-ε (C/EBPε) gene. To date, two patients have been reported transcription factors that regulate proliferation, differentiation, and by guest on October 1, 2021 who carry C/EBPε frameshift mutations that result in abrogated apoptosis in a variety of cell types (5, 6). The C/EBP family consists protein expression (2, 3). In addition, another patient with SGD had of six members (C/EBPa, b, g, d, ε,andz), and cellular expression a heterozygous missense mutation of the C/EBPε gene, but this of each C/EBP is tightly regulated. They bind to DNA through the mutation was unlikely to have caused disease because it resulted highly conserved basic leucine zipper (bZIP) domain. C/EBPε is restricted to granulocytes and is essential for their terminal differ- entiation (6, 7). C/EBPε transcription primarily occurs at the mye- *Department of Pediatrics, Institute of Medical, Pharmaceutical and Health Sci- locyte–metamyelocyte stage of differentiation and decreases in ences, School of Medicine, Kanazawa University, Kanazawa 920-8641, Japan; polymorphonuclear neutrophils. The human C/EBPε gene produces †Department of Stem Cell Biology, Institute of Medical, Pharmaceutical and Health Sciences, School of Medicine, Kanazawa University, Kanazawa 920-8640, Japan; four isoforms of 32, 30, 27, and 14 kDa that are functionally dif- ‡Department of Dermatology, Komatsu Municipal Hospital, Komatsu 923-0961, ferent; only the 32-kDa C/EBPε has full transactivating potential (8– Japan; xDepartment of Infection and Host Defense, Shinshu University Graduate School { 10). C/EBPε is indispensable for expression of genes encoding of Medicine, Matsumoto 390-8621, Japan; Division of Hematology and Oncology, Cedars-Sinai Medical Center, University of California Los Angeles School of Med- proteins that reside in specific granules of neutrophils, such as lac- icine, Los Angeles, CA 90048; and ‖Cancer Science Institute of Singapore, National toferrin and defensins. Many features of SGD are manifested by University of Singapore, Singapore 117599, Singapore C/EBPε-deficient mice in which neutrophils are morphologically and 1 T.W. and T.A. contributed equally to this work. functionally abnormal and eosinophil numbers are decreased (8). Received for publication September 8, 2014. Accepted for publication May 4, 2015. In this study, we describe another case of SGD with a novel 2-aa This work was supported by a Grant-in-Aid for Scientific Research from the Ministry deletion in the bZIP domain of C/EBPε, and we report the mech- of Education, Culture, Sports, Science and Technology of Japan and a grant from the anism that leads to SGD. We also completed clinical, cellular, and Ministry of Health, Labor, and Welfare of Japan, Tokyo. molecular comparisons for this patient and the previously reported Address correspondence to Dr Taizo Wada, Department of Pediatrics, School of Medicine, Institute of Medical, Pharmaceutical and Health Sciences, Kanazawa Uni- two cases of SGD (2, 3), and we discuss the functional significance versity, 13-1 Takaramachi, Kanazawa 920-8641, Japan. E-mail address: taizo@staff. of the mutation. kanazawa-u.ac.jp The online version of this article contains supplemental material. Abbreviations used in this article: bZIP, basic leucine zipper; C/EBPε, CCAAT/enhancer Materials and Methods binding protein-ε; Gata1, GATA binding protein 1; MaBP, major basic protein; MBP, Patients maltose-binding protein; NGAL, neutrophil gelatinase-associated lipocalin; Prg2, pro- teoglycan 2; SGD, neutrophil-specific granule deficiency; WT, wild-type. We studied two Japanese patients with SGD. Patient P1 is a 55-y-old woman who has suffered since late infancy from recurrent skin infections that often Copyright Ó 2015 by The American Association of Immunologists, Inc. 0022-1767/15/$25.00 required more than 2 mo to heal. After hospitalization because of severe www.jimmunol.org/cgi/doi/10.4049/jimmunol.1402222 The Journal of Immunology 81 otitis media at 54 y of age, she was referred to our hospital for suspec- Biotin-labeled DNA pull-down assay, MBP pull-down assay, ted immunodeficiency. A history of parental consanguinity appeared likely. and Western blot analysis However, her father had already died of a heart attack, and her mother refused genetic analysis. Her elder brother had similar skin symptoms and died of Biotin-labeled DNA pull-down assay was performed as described previ- enterocolitis at 10 y of age. Another brother also died early after birth from ously (13, 17). Briefly, biotin-labeled oligonucleotides containing the human unspecified causes. Two children of patient P1 and her granddaughter were lactoferrin gene C/EBPε binding site (59-GGGTGTCTATTGGGCAACA- healthy, and they did not want genetic testing. Clinical and genetic data of GGGCGGG-39) were incubated with cell extracts from HEK293 cells patient P2 have already been published (3, 11). Patient P2 is now 40 y old. transfected with either pCAGIP-Myc-C/EBPε or its mutant counterparts Approval for the study was obtained from the Human Research Committee (DRS, del5bp, and insA) in the presence of streptavidin-agarose (Novagen, of Kanazawa University
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